1. Technical Field that Invention Belongs
The present invention relates to a microscope, particularly, a new optical microscope with high performance and high function so as to obtain a high spatial resolution by illuminating dyed sample with lights of plural wavelengths from a high laser light source of high functionality.
2. Description of Related Technology
The technology of the optical microscope is known at early time, and the microscope of various types has been developed. Moreover, in recent years, the microscope system with high performance has been developed more due to the advancement of the peripheral technology including the laser technology and the electronic images technology.
In such a background, a microscope with high function, capable of controlling the contrast of the obtained image and capable of performing chemical analysis, has been proposed, by the use of the double resonance absorption process generated by illuminating the sample with lights of plural wavelengths (For example, refer to Japanese Patent Application Opened No. 184,552/1996).
This microscope selects a specific molecule by using the double resonance absorption, and observes absorption and fluorescence caused by a specific optical transition. This principle is explained referring to FIGS. 11–14. FIG. 11 shows the electronic structure of the valence electron orbital of the molecule constructing a sample, and first of all, the electron of the valence electron orbital of the molecule in a ground-state (S0 state) shown in FIG. 11, is excited by the light of wavelength λ1, thereby assuming the first electron excited state shown in FIG. 12 (S1 state). Next, the electron of the valence electron orbital is excited by the light of another wavelength λ2, thereby assuming the second electron excited state shown in FIG. 13 (S2 state). By this excited state, the molecule emits fluorescence light or phosphorescence light, and then returns to the ground-state as shown in FIG. 14.
In the microscopy using the double resonance absorption process, the absorption image and the luminescence image are observed by using the absorption process shown in FIG. 12 and the luminescence of fluorescence and the phosphorescence shown in FIG. 14. In this microscopy, firstly, the molecule constructing the sample with the light of resonant wavelength λ1, as shown in FIG. 12, is made to be excited in S1 state by the laser light etc. But in this case, the number of molecule in S1 state within the unit volume increases as intensity of the irradiated light increases.
Here, the linear absorption coefficient is given by the product of the absorption cross section per one molecule and number of molecules per unit volume, so that in the excitation process shown in FIG. 13, the linear absorption coefficient to resonance wavelength λ2 of continuously irradiated light, depends on an intensity of firstly irradiated light with wavelength λ1. That is, the linear absorption coefficient to wavelength λ2 can be controlled by intensity of the light with wavelength λ1. This means that if the sample is irradiated by the lights of two wavelengths of wavelength λ1 and wavelength λ2, and the transmitted image due to the wavelength λ2 is obtained, the contrast of the transmitted image can be completely controlled by the light with the wavelength λ1.
Moreover, when the de-excitation process due to the fluorescence or phosphorescence in the excited state shown in FIG. 13 can be realized, the emission intensity is proportional to the number of molecules in S1 state. Therefore, the contrast of the image can be controlled even in the case of utilization as the fluorescence microscope.
In addition, in the microscopy using the double resonance absorption process, not only the control of the image contrast, but also the chemical analysis can be realized. That is, the outermost shell valence electron orbital shown in FIG. 11 has an energy level inherent to each molecule, so that the wavelength λ1 is different according to the molecule, at the same time, the wavelength λ2 becomes also inherent in molecule.
Here, even in case of the illumination by the single wavelength in conventional microscope, the absorption image or the fluorescence imaging of the molecule specified to some degree can be observed, but in general, the wavelength regions of the absorption band in some molecules are overlapped, so that the accurate identification of the chemical composition of the sample can not be performed.
On the contrary, in the microscopy using the double resonance absorption process, the molecule absorbed or emitted by two wavelengths of the wavelength λ1 and the wavelength λ2, is strictly specific, so that the chemical composition of the sample can be identified more accurately than conventional microscopy. Moreover, in case of exciting the valence election, only the light with a specified electric field vector for molecular axis, is absorbed strongly, so that when the polarizing direction of the wavelength λ1 and the wavelength λ2 are decided and a picture of the absorption image or the fluorescence imaging is obtained, even the identification of the direction of the orientation can be possible as for the same molecule.
Moreover, in recent years, the fluorescence microscope with a high spatial resolution beyond the diffraction limit by using the double resonance absorption process, is also proposed (For example, refer to Japanese Patent Application Opened No. 100,102/1997).
FIG. 15 is a conceptual diagram of the double resonance absorption process in the molecule, and shows the states that the molecule in ground-state S0 is excited to first electronic excited state S1 with the light of wavelength λ1, and is excited to the second electronic excited state S2 with the light of wavelength λ2. Moreover, FIG. 15 shows that fluorescence from the second electron excited state S2 of a certain kind of molecule is extremely weak.
A very interesting phenomenon occurs in the case of the molecule with the optical property shown in FIG. 15. FIG. 16 is a conceptual diagram of the double resonance absorption process in the same manner as FIG. 15, X axis as abscissa shows an extension of spatial distance, and shows a spatial area A1 irradiated by the light with wavelength λ2 and a spatial domain A0 not irradiated by the light with wavelength λ2.
In FIG. 16, a lot of molecules in electron excited state S1 are generated by the excitation of the light with wavelength λ1 in spatial domain A0, in this case, the fluorescence emitted with wavelength λ3 from the spatial area A0, is seen. However, the light with wavelength λ2 is irradiated in the spatial area A1, so that almost molecules of electron excited state S1 are immediately excited in the electron excited state S2 of high order, and thus the molecule in electron excited state S1 does not exist. Such a phenomenon is confirmed by some molecules. As a result, in the spatial area A1, the fluorescence of wavelength λ3 is disappear completely, and also the fluorescence from the electron excited state S2 does not exist originally so that the fluorescence is completely controlled in the spatial domain A1 (fluorescent suppression effect) and thus the fluorescence will be emitted only from the spatial area A0.
This has an extremely important meaning on considering from the field of application in the microscope. That is, in the conventional scanning laser microscope etc., the laser beam is focused into a micro-beam by the condenser lens and scans on the sample to be observed, but in that case, the size of the micro-beam becomes a diffraction limit decided by the numerical aperture of the condenser lens and the wavelength of the beam, and thus a spatial resolution better than this limit can not fundamentally be expected.
However, in the case of FIG. 16, by overlapping two kinds of lights with the wavelength λ1 and the wavelength λ2 spatially and proficiently, and then controlling the fluorescent region by the irradiation of the light with the wavelength λ2, for example, on paying attention to the irradiation area of the light with the wavelength λ1, a fluorescent region can be narrowed more than the diffraction limit decided by the numerical aperture and the wavelength of the condenser lens, and thus the spatial resolution can be substantially improved. Therefore, by utilizing this principle, a super-resolution microscope, for example fluorescent microscope, that uses the double resonance absorption process better than the diffraction limit, can be achieved.
In addition, the signal to noise ratio can be improved by adjusting the irradiation timing of two lights with the wavelength λ1 and the wavelength λ2, and the conditions, that effectively causes fluorescent controlling, are proposed, too, as a result, the super-resolution can be developed more effectively (For example, refer to Japanese Patent Application Opened No. 95,120/1999).
To concrete example of such a super-resolution microscopy, it has been proposed that the light (particularly laser beam) with the wavelength λ1 by which a fluorescent labeler molecule is excited from the ground-state S0 to the electron excited state S1, is assumed to be a pump light, the light with the wavelength λ2 by which the fluorescent labeler molecule is excited from the electron excited state S1 to the electron excited state S2 is assumed to be an erase light, as shown in FIG. 17, the pump light is made radiated from a light source 81, and the erase light is made radiated from a light source 82, respectively, after reflecting on a dichroic mirror 83, the pump light is made focused on a sample 85 by a beam-condensing optical system 84, and after shaping into a hollow beam by a phase plate 86, the erase light is transmitted through the dichroic mirror 83, whereby it spatially overlapped with the pump light. Then, the erase light is focused onto the sample 85 by the beam-condensing optical system 84. (For example, refer to Japanese Patent Application Opened No. 272,340/1997).
According to this microscope, fluorescence intensity from except for the near the optical axis region in which the intensity of the erase light becomes 0, are controlled, so that as a result, only a fluorescent labeler molecule, that exists in a region narrower than the spot size of the pump light, (Δ<0.61·λ1/NA; NA is a numerical aperture of the condensing optical system 84) is observed, and thus the super-resolution is achieved.
Moreover, the phase plate 86 for making the erase light to a hollow beam is generated in such a manner that for example, as shown in FIG. 18, the phase difference π is given in at the point symmetrical position to the optical axis. Furthermore, an optical spatial modulator using a liquid crystal plane, or deformable mirror for controlling the shape of the mirror itself with accuracy of the wavelength order, can also be used.
However, it is found that according to the examination of various experiments given by the present inventor etc., the point to be improved as explained hereinafter exists in the conventional super-resolution fluorescent microscope.
Hereafter, the case that a super-resolution fluorescent microscope is constructed as shown in FIG. 19, is explained as an example. The super-resolution fluorescent microscope shown in FIG. 19 is disclosed in Japanese Patent Application Opened No.100,102/2001, and the inventors observe a sample 100 dyed by rhodamine 6G. In this microscope, the laser beam with the wavelength 1064 nm generated from Nd:YAG laser 101 of the mode-locking type is wavelength-converted into a laser beam with a wavelength 532 nm of the second harmonics by a wavelength conversion element 102 consists of β-BaB2O4(BBO) crystal, the laser beam is divided into two optical paths of transmitted light and the reflected light by a half mirror 103, the transmitted light is used as a pump light. After transmitting through dichroic mirrors 104 and 105, the transmitted light is made focused on the sample 100 put on two dimensional moving stage 107 by an objective lens 106.
Moreover, after reflected on a reflection mirror 108, the reflected light due to the half mirror 103 is wavelength-converted into the laser beam with the wavelength 599 nm by a Raman shifter 109 consisting of Ba(NO3)2 crystal. After reflected as an erase light by a reflection mirror 110, the laser beam is shaped to the hollow beam by a phase plate 111, in addition, is aligned with the dichroic mirror 104 on the same axis as the pump light, and thus is made focused on to the sample 100 through the dichroic mirror 105 by the objective lens 106.
On the other hand, after being reflected on the dichroic mirror 105 through the objective lens 106, the fluorescence emitted from the sample 100 is received on a photo multiplier 115 through a fluorescent condenser lens 112, a sharp cut filter 113 and a pin hall 114.
In such a construction of the super-resolution fluorescent microscope, the super-resolution of developing precision is influenced a great deal by the beam shape of the erase light. That is, if an ideal hollow beam is not formed, it is feared that the spatial resolution decreases oppositely, the fluorescence signal decreases, and thus the signal to noise ratio decreases.
Here, the shaping accuracy of the hollow beam depends on the phase modulation technique, that is, the precision of a wavefront control technique. For example, when the wavefront of the erase light falls into disorder by some causes, the shape of the hollow beam crumbles, and the counterbalance of the electric field intensity at a central portion of optical axis becomes imperfect and the electric field intensity remains. Therefore, under such conditions, when the pump light and the erase light are overlapped and focused onto the sample, the intensity of fluorescence is controlled even at the central portion of optical axis, and thus the spatial resolution is not only decreased, but also the signal to noise ratio is decreased.
The cause of the disorder of the wavefront is variously thought, but there are, for example, a distortion of wavefront of laser beam, an error of shape accuracy of respective optical elements, through which the laser beam passes, and an error of the alignment.